[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May 04, 2022 to July 28, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

adopted 30 June 2022

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Justification for non-LLNA method:

The correlation of upregulation of immunologically relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event of the skin sensitisation process as described by the AOP for skin sensitisation. This method, which measures the markers of DC activation, using the DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals. Therefore, this test method is considered to be able to detect chemicals that cause skin sensitisation when used within Integrated Approaches to Testing and Assessment (IATA), together with other relevant complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as non-testing methods, including read-across from chemical analogues. Depending on the regulatory framework, positive results generated with these methods may be used on their own to classify a chemical into UN GHS category 1.

Test material

Test material information

Constituent 1

Reference substance name:

Phosphonic acid, (4-morpholinylmethylene)bis-, tetraethyl ester

Cas Number:

59646-46-7

Molecular formula:

C13H29NO7P2

IUPAC Name:

Phosphonic acid, (4-morpholinylmethylene)bis-, tetraethyl ester

Test material form:

liquid: viscous

In vitro test system

Details of test system:

THP-1 cell line [442E]

Details on the study design:

Preparation of the Test Item

The test item was freshly prepared immediately prior to use.
The test item was dissolved in 0.9% NaCl solution at a concentration of 500 mg/mL. Vortex mixing was used to aid solubilisation. No correction factor for the purity was used.
The working stock solutions were prepared by diluting each stock solution 50 times with cell culture medium. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent (0.9% NaCl solution) was present at a constant volume ratio of 1% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Test System

FACS
FACS: BD FACSLyric
Software: BD FACSuite 1.2.1

Cell line
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 106 cells/mL.
Cells were cultured in 175 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 µg/mL streptomycin in a humidified incubator at 37 +- 1°C and 5% CO2.

Dose Groups
1. Medium Control: cell culture medium
2. Solvent Control: cell culture medium
0.2% DMSO (v/v) in cell culture medium
3. Positive Control: 4 µg/mL DNCB
4. Test Item: 8 concentrations of the test item
(dose finding assay/ main experiment)
dose finding assay 1:
5000, 2500, 1250, 625, 312.5, 156.25, 78.13 and 39.06 µg/mL
main experiment 1 and 2:
5000.00, 4166.67, 3472.22, 2893.52, 2411.27, 2009.39, 1674.49 and 1395.41 µg/mL

Pre-Experiments

Reactivity Check of the Cells Stock
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. For the reactivity check of the cell batch additional negative and positive controls were included. DNCB CAS No. 97-00-7, ≥ 99% purity) at a final concentration of 4 µg/mL and nickel sulphate (CAS No. 10101-97-0, 99% purity) at a final concentration of 100 µg/mL served as positive control while lactic acid (CAS No. 50-21-5, ≥ 99% purity) at a final concentration of 1000 µg/mL served as negative control. Cells were accepted when both, DNCB and nickel sulphate produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.

Solvent Finding
Solubility of the test item was determined prior to the dose-finding assay experiment. The test item was soluble in 0.9% NaCl at a final concentration of 500 mg/mL.

Experimental Procedure

Dose Finding Assay
Starting from 500 mg/mL solutions of the test chemical, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 50-fold into culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution
For testing, THP-1 cells were pre-cultured for 48 h -72 h in culture flasks at a cell density of
0.1 – 0.2 x 106 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 106 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 106 cells/well).
The solvent controls and the test item working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at
37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer). After washing, cells were re-suspended in 600 µL FACS buffer.
200 µL of the cell suspension was transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 µg/mL.
The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration.
The CV75 value, i.e. the concentration showing 75% cell survival, was calculated by log-linear interpolation utilizing equation 10 2. The CV75 value was used to calculate the concentration range of the test item for the main experiment.
 
10.7.2. CD54 and CD86 Expression
The test item was dissolved using 0.9% NaCl as determined in the pre-experiment. As the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item (500 mg/mL) was used as starting dose.
The test item was diluted to a concentration of 500 mg/mL and then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 50-fold into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor.
For testing, THP-1 cells were pre-cultured for 48 h – 72 in culture flasks at a cell density of
0.1 – 0.2 x 106 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 106 cells/mL. Then, 500 µL of the cell suspension was seeded into a 24 well flat-bottom plate (1 x 106 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at
37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer in an appropriate manner. After washing with FACS buffer twice, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 µg/mL).
The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated. The cell viability was calculated.

Data Analysis
FACS data analysis was performed using the software BD FACSuite 1.2.1. Further data analysis like calculation of the CV75, calculation of the RFI and calculation of the Effective Concentration 150 (EC150) and Effective Concentration 200 (EC200) values were performed using the software Microsoft Excel 2010. The mean values and standard deviations of the single replicates were determined using the respective excel commands.


Evaluation of the Results
Prediction Model
For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day or on the same day provided that for each run: independent fresh stock solutions and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used.
Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs. In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 µg/mL for 0.9% NaCl solution; 1000 µg/mL for DMSO or a different organic solvent) even if the cell viability is >90%.
A negative result for test items with a Log KOW > 3.5 should be considered as inconclusive.

Vehicle / solvent control:

other: Since the test item was solubilized in either cell culture medium or 0.9% NaCl, the medium control served as solvent control. Since the positive control was solubilized in DMSO, a DMSO control was included and served as solvent control for the PC

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Results and discussion

Positive control results:

The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

RFI CD86>200 [442E]

Value:

0 %

Cell viability:

Relative cell viability at the highest test item concentration was reduced to 95.3% (CD86).

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

RFI CD86>200 [442E]

Value:

0 %

Cell viability:

Relative cell viability at the highest test item concentration was reduced to 95.7% (CD86)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

RFI CD54>150 [442E]

Value:

0 %

Cell viability:

Relative cell viability at the highest test item concentration was reduced to 95.2%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

RFI CD54>150 [442E]

Value:

0 %

Cell viability:

Relative cell viability at the highest test item concentration was reduced to 92.8% (CD54).

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experimental runs. Therefore, the test item can be considered not to activate dendritic cells, the third key event in the skin-sensitization AOP.
The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of Integrated Approach to Testing and Assessment (IATA) in combination with other complementary information e.g. derived from in vitro assays addressing other key events of the skin-sensitisation AOP.

Executive summary:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study, the test item was dissolved in 0.9% NaCl. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 mg/mL (final concentration in the test media 5000 µg/mL to 39.06 µg/mL) were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:

5000, 4166.67, 3472.22, 2893.52, 2411.27, 2009.39, 1674.49 and 1395.41 µg/mL

In all experiments no precipitation and no turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 95.7% (CD86), 92.8% (CD54) and 94.6% (isotype IgG1 control) in the first experiment and to 95.3% (CD86), 95.2% (CD54) and 92.9% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

Therefore, under the conditions of this study the test item was negative for dendritic cell activation.